The preparation of derivatives of certain substances makes it possible to determine small amounts of these in complex samples such as blood plasma and urine. Chiral compounds can furthermore be resolved by formation of diastereomers with chiral reagents followed by separation using conventional HPLC-columns (high performance liquid chromatography) or GC-columns (gas chromatography).
Separation and determination of compounds such as amines, alcohols and amino acids and optical isomers of these has become more and more important because of their biochemical and pharmaceutical interest. A large number of reagents for derivatization of amines and amino acids for subsequent separation, for example by means of liquid chromatography, have been suggested but just a few have found any extensive use. Among the achiral reagents 9--fluorenylmethyl chloroformate (FMOC) has been developed during the last few years and found use for derivatization of amino acids. The amino acid derivatives are separated by HPLC and then determined by fluorescence detection, cf J. Chromatogr. 1983, pages 609-618 (S. Einarsson, B. Josefsson, S. Lagerkvist). Fluorescence detection with these reagents give detection at very low concentrations while UV-detection is limited to higher concentrations (low fmole respectively low pmole).
Among the chiral derivatization reagents, i.e. reagents which can be used also for separation of optical isomers, the reagent (+)-1-(9-fluorenyl)ethyl chloroformate (FLEC) has recently been developed and is disclosed in WO 87/06929. Also for this reagent the fluorescence detection is very good while the UV-detection is limited to higher concentrations.
The demands on and the desires for a reagent for separation purposes according to what has been discussed above are to a great extent the same for both achiral and chiral reagents. In addition to the fact that it of course should be possible to prepare them in a satisfactory manner, the reagents should form stable derivatives of the compounds to be determined and/or separated, in the first place with compounds containing amino groups, and hereby preferably both with primary and secondary such groups, but of course also with as many other types of compounds as possible for increased universality in use. The detection sensitivity should be as high as possible and it is highly desirable that more than one detection method can be used. It shall be possible to separate the prepared derivatives by conventional column separation. For chiral derivatization reagents it is also extremely important that it is possible that they can be prepared optically pure and that the derivatization can be carried out under mild conditions to avoid risk of racemization.